Deoxyuridine triphosphate nucleotidohydrolase (dUTPase, E.C. 3.6.1.23) is an ubiquitous enzyme which hydrolyzes deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP) and pyrophosphate, typically in the presence of magnesium ions. This reaction is thought to occur primarily to limit pools of intracellular dUTP in order to prevent significant uridine incorporation into DNA during replication and repair. A second role of dUTPase is to provide substrate (dUMP) for the de novo synthesis of thymidylate.
Two groups of researchers, McIntosh et al., PNAS, 89:8020-8024 (1992) and Strahler et al., PNAS, 90:4991-4995 (1993), have reportedly isolated the human dUTPase enzyme and characterized the enzyme by its cDNA and amino acid sequences.
McIntosh reported a cDNA of 526 base pairs containing an ORF which encoded a protein of 141 amino acids and a 3f flanking sequence following the ORF. Strahler reported the identical cDNA and amino acid sequence as did McIntosh, with the exception of two additional bases at the 51 end of the cDNA and a longer 3f flanking sequence. The human dUTPase reported by both groups was found to have a high degree of homology with dUTPase from other organisms including that from yeasts, bacteria and viruses. Strahler further reported that human dUTPase exists in both, phosphorylated and a non-phosphorylated forms.
International patent application no WO97/36916 discloses the sequence of nuclear and mitochondrial isoforms of dUTPase.
In both prokaryotic and eukaryotic cell systems, dUTPase has been clearly shown to be an essential enzyme, without which the cell will die. Lack of dUTPase leads to elevated cellular dUTP pools, resulting in an increased misincorporation of uridine into DNA. In addition to prokaryotes and eukaryotes, a number of viruses, such as herpes simplex, are known to encode a dUTPase function.
International patent application no WO95/15332 proposes a range of uridine di- and triphosphate analogues in which the oxygen atoms between phosphate groups are replaced with methylene, secondary amine or tertiary amine, and/or oxo functions on the phosphate are replaced with sulphur. These compounds are postulated as cytostatics for use against rapidly growing cancer cells and/or antivirals against herpes. Substantially similar compounds are disclosed in Zalud et al Adv. Exp. Med. Biol. 1995 370 135-138 and Persson et al Bioiorg Med Biochem 1996 4 553-556. It should be noted, however that these compounds have been primarily designed for crystallographic purposes and the analysis of enzyme kinetics. These compounds therefore do not possess physicochemical attributes suggestive of a drug.
The present inventors have established that the substrate specificity of the dUTPases of certain protozoal and bacterial parasites of man differ from the corresponding human cellular and mitochondrial enzymes to such an extent that a specific set of inhibitor compounds can be prepared which selectively inhibit the parasite dUTPase without substantially inhibiting the human counterparts. Examples of such parasites include Plasmodium species especially P. falciparum responsible for malaria, Mycobacterial species, especially M tuberculosum responsible for tuberculosis and Leishmania spp.
Hidalgo-Zarco and González-Pacanowska Current Protein and Peptide Science, 2001, 2, 389-397 describe the isolation and characterisation of trypanosomal dUTPases. In contrast to the trimeric form of dUTPase shared by human and malarial enzymes, the trypanosomal enzyme is a dimmer. Competitive inhibition of Leishmania dUTPase was shown by the triphosphate substrate analogue α-β-imido-dUTP, whereas no inhibition of that parasite was apparent in the case of 5′-O-(4-4′-dimethoxytrityl)-2′-deoxyuridine.